The invention relates to methods for labeling nucleotides using linkers (linking moieties between labels and bio-organic molecules, which linkers are based on platinum compounds).
Platinum (coordination) compounds have been considered interesting molecules for a very long time. For a review of these compounds and their uses we refer to Reedijk et al. (Structure and Bonding 67, p.53-89, 1987). Especially Cis-platinum has received a lot of attention as a possible anti-tumour drug. This anti-tumour reactivity of platinum compounds originates from their having at least two reactive groups (preferably cis-oriented towards each other), which make it possible to cross-link DNA molecules, thereby inhibiting the replication of these DNA molecules.
The British patent application 2 148 891 discloses cis-platinum complexes, which are six-coordinated. The platinum is attached to two halogens or hydroxy groups, two additional halogens and to an ethylene diamine derived group, such as 1,2-diamino-2-methylpropane or 1,2-diamino-2-methylbutane. The complexes are said to have an excellent anti-tumor effect.
In the European patent application four-coordinated complexes of platinum to 2,3-alkyl-1,4-butanediamine and two halogens are described for their anti-tumor effect.
Different four-coordinated platinum complexes are described in the European patent application 0 386 243. The complexes comprise a diamine bidentate ligand and two 2-arylalkanoic acid or 3-aryl-2-oxoalkanoic acid ligands. These complexes are stated to have a strong growth inhibiting action on certain leukemia cells and are used for their oncostatic activity.
U.S. Pat. No. 4,207,416 discloses ethylenediamine-platinum(II) 2,4-dioxopyrimidine complexes as having a high anti-tumor activity and low mammalian toxicity.
A different use of platinum (coordination) compounds has been disclosed in PCT application (WO92/01699) wherein a platinum compound having only two reactive moieties (denominated as leaving groups therein) is reacted with a fluorescein to obtain a labeled platinum compound which can bind (non-covalently) to a nucleic acid, preferably at the N-7 position of a guanine residue.
Several methods for labeling nucleotides have been described in the literature. For a long time, the standard method has been to use radioactive isotope labeling. However, there are a number of problems associated with the use of radioisotopes, such as potential health hazards, disposal problems and instability problems.
In order to overcome these problems, Dale et al., Biochemistry, 14, (1975), 2447-2457, have proposed to use direct covalent mercuration as a labeling technique for nucleotides and polynucleotides. It was found, that cytosine and uracil may be mercurated at their C5-position under mild conditions. Further, Gebeyehu et al., Nucleic Acids Research, 15, (1987), 4513-4534, have reported that adenine and cytosine may be labeled with biotin derivatives through an aliphatic linker of from 3 to 17 atoms.
A major drawback of these known methods is that they are not suitable for labeling all different nucleotides. For instance, Dale et al. reported that their covalent mercuration method leads to negative results for adenine, thymine and guanine bases. In some cases, for example when only a few residues of a certain nucleotide are present in a certain nucleic acid or when the terminating nucleotide residue of a nucleic acid has to be labeled, it is desired to have at one""s disposal a method for labeling any nucleotide residue.
The present invention provides such a method. The method for labeling nucleotides of the invention comprises the steps of:
reacting a reactive moiety of a linker of the formula 
wherein X represents any stabilizing bridge and wherein A and B represent the same or different reactive moieties, with an electron donating moiety of a spacer, which spacer comprises a chain having at least four atoms and at least one heteroatom in the chain, which spacer further comprises said electron donating moiety at one end of the chain and a reactive moiety at the other end of the chain; reacting the reactive moiety of said spacer with a label; reacting the other reactive moiety of said linker with a nucleotide.
According to the invention, the linker may first be attached to the nucleotide and then to the spacer, or vice versa and the spacer may first be coupled to the label and then to the linker or vice versa.
The reactive moiety of the spacer may be any reactive moiety that will enable the reaction between the spacer and the label in such a manner that a labeling moiety comprising a label and a spacer is formed, which labeling moiety is sufficiently stable.
The main purpose for labeling nucleotides is that these labeled nucleotides can be incorporated in nucleic acid molecules. Modified nucleotides, especially those wherein a (bulky) label is attached to the nucleotide, are often built-in into nucleic acids with a much lower efficiency. The methods according to the invention result in labeled nucleotides which are built-in into nucleic acids with a higher efficiency than the labeled nucleotides available to date. This is probably for due to the selection of the spacers according to the invention in combination with the platinum-based linkers according to the invention.
The label to be used according to the invention is not critical. In principle all labels which can be attached to a nucleotide and are employed to date can be used. These labels may be radioactive labels, enzymes (which need reaction with a substrate to be detected), specific binding pairs components such as avidin, streptavidin or biotin, biocytin, iminobiotin, colloidal dye substances, fluorochromes (rhodamin, etc.), reducing substances (eosin, erythrosin, etc.), (coloured) latex sols, digoxigenin, metals (ruthenium), metal sols or other particulate sols (selenium, carbon and the like), dansyl lysin, Infra Red Dyes, coumarines (amino methyl coumarine), antibodies, protein A, protein G, etc. The invention has most benefits with bulkier labels such as biotin, avidin, streptavidin, digoxygenin or a functional equivalent thereof.
The invention is not limited to nucleotides or nucleosides as such; derivatives and functional equivalents are also included. The usual nucleotides adenine, thymidine, cytosine, guanine and uridine are preferred. Especially the purines are preferred which have a very good incorporation rate.
For coupling of the spacer to the platinum linker an electron donating moiety is required. In a preferred method the electron donating moiety is an amine or a thiolate anion, which have both proven to be very succesful. It was found that aromatic amines, such as imidazoles or purines, are capable of forming very strong bonds to platinum and thus are very suitable for use as the electron donating moiety.
The spacer is an important aspect of the present invention; it provides the easiest coupling between label and linker. For avoiding steric hindrance in incorporation of the nucleotide into the nucleic acid it should at least be four atoms long, preferably it is at least four carbon atoms long and has at least one heteroatom in that carbon chain. A heteroatom confers a certain amount of rigidity on the spacer. This rigidity provides an additional assurance that steric factors will not obstruct a convenient linking of a nucleotide and a label. It is preferred that at least one heteroatom is an oxygen atom, which positively effects the hydrophilicity of the spacer.
Preferably, the spacer comprises no more than 20 carbon atoms in the chain, which is preferably an essentially non-branched chain, thus causing no steric hindrance. The reason for this will be clear.
A highly preferred spacer is 1,8-diamino-3,6-dioxaoctane, herein referred to as Dadoo. Dadoo is a very flexible compound with a distal primary amine group and a size that makes it very suitable for use as spacer according to the invention.
Another highly preferred spacer of the invention is an oligolysine or a polylysine. Due to their structure and conformation, these molecules create the most convenient environment for an optimal interaction among the actual label, the nucelotide and the platinum. An additional advantage of the use of lysine chains as the spacer is, that by altering the number of lysine units in the chain, the optimal conditions for specific labels and nucleotides or nucleic acids can be attained. Given a certain application, the skilled person will easily determine how many lysine units are required for optimum results.
An especially interesting labeling moiety comprising a label and a spacer, has the formula 
or the formula 
wherein X represents any stabilizing bridge, Z and Zxe2x80x2 represent a non-leaving ligand and n is an integer of from 2 to 10.
Accordingly, the linker-spacer-label-system, or labeling substance, with the labeling moiety of formula (II) or formula (III) has the formula 
or the formula 
wherein A, X, Z, Zxe2x80x2 and n have the above meanings.
The non-leaving ligands Z and/or Zxe2x80x2 are preferably an NH3, NH2R, NHR2 or NR3 group, wherein R represents an alkyl group having from 1 to 6 carbon atoms, because these ligands have an even smaller leaving-group character than other non-leaving ligands.
The interesting feature of using the labeling moieties having formulas (II) and (III) is that both the nucleotide and the actual label have the benefit of being bonded directly to a platinum atom, while at the same time these moieties are sufficiently far apart to avoid steric hindrance.
The linkers according to the invention preferably are platinum compounds wherein X represents an aliphatic diamine. In a preferred embodiment of the invention, one or both of the nitrogen atoms of the aliphatic diamine are shielded. A suitable manner of shielding these nitrogen atoms consists of substitution with one or two alkyl groups of from 1 to 6 carbon atoms, preferably methyl groups. This is advantageous in that hydrogen bonding between the triphosphate group of a nucleotide and the stabilizing bridge is prevented. Preferably, a diamine having 2-6 carbon atoms is use, most preferably an ethylene diamine group, which is well-known for its stabilizing effect on this class of platinum compounds. In this case, the linker has the formula 
wherein G represents hydrogen or an alkyl group of from 1 to 6 carbon atoms and A and B represent the same or different reactive moieties.
The coupling or reactive moieties A and B are preferably the same and selected from the group consisting of NO3xe2x88x92, SO3xe2x88x92, Clxe2x88x92, Ixe2x88x92, or other halogens.
The invention of course also encompasses a labeled nucleotide obtainable by a method as disclosed above.
In addition, the invention encompasses a labeling substance for labeling nucleotides by a method as disclosed above. The labeling substance of the invention has the formula 
wherein X and A have the above meanings and the labeling moiety comprises a label and a spacer as described above. Of course the labeling substances of the invention can also be used for labeling purposes other than labeling nucleotides. It was found that numerous (bio-) organic compounds, i.e. nearly every bio-organic molecule which contains an accessible sulphur or nitrogen atom, for example proteins, can be labeled with the platinum compounds of the invention.
A great advantage of the invention arises from the use of the platinum compounds having formula (I) as linkers in the methods of preparing labeled nucleotides according to the invention. These linkers can be prepared by very convenient and reliable methods.
In WO92/01699 the starting compounds disclosed for preparing labeled platinum compounds are platinum(II)(ethylenediamine)dichloride and platinum(II)(ethylenediamine)(Me2SO)Cl. The first one can be obtained commercially, the second one (the preferred one) must be synthesized. In the dichloride compound, the Cl-ions are less readily substituted by a label or a nucleotide, respectively. In the latter case, the total nucleotide labeling time will be appreciably longer, up to several hours, instead of several minutes.
The methods for preparing the linkers that are used in the method of labeling nucleotides according to the invention are based on the selection of suitable starting compounds of the formula PtE4 wherein E is an electronegative group, preferably a halogen or NO3xe2x88x92 or SO3xe2x88x92. The reaction, which is described in the examples, of these starting compounds with e.g. ethylenediamine is very simple and efficient. Moreover, this reaction leads to very suitable symmetric intermediate compounds for producing labeled nucleotides. A major advantage of using these compounds is that when a stabilizing bridge for the resulting platinum compound has to be attached that no blocking reagents have to be employed. Another advantage is that the resulting intermediate compounds can again be labeled without the use of blocking agents. Therefore steps removing blocking agents can be eliminated completely. Furthermore the yields of these reactions are very high. Yet another advantage of the use of these symmetrical starting compounds is that no mixtures of different resulting compounds can be formed, which may interfere with the following reaction and reduce yield or require extra separation steps.
A very suitable intermediate compound according to the invention is platinum(II)(ethylenediamine)(NO3)2. This substance can very easily be provided with a suitable spacer and a labeling group, resulting in labeling substances which can, through substitution of the remaining NO3-group be linked to a nucleotide quite easily. Furthermore the methods for producing these compounds and the resulting compounds do not involve highly toxic substances such as DMSO.
The intermediate compounds can be labeled with any suitable label (also known as marker) through a spacer as disclosed hereinabove.
Furthermore, the known advantages (from WO92/01699 for instance) are of course also obtained with the present methods and compounds. Another advantage of the platinum compounds is that they can be detected more or less directly by using the platinum as a nucleus for depositing silver or other metal crystals.
By binding the labeling substance to a nucleotide residue, DNA or RNA molecules, be it single stranded or otherwise, can be easily detected, but it also allows for the production of probes for hybridization techniques wherein unlabeled DNA/RNA molecules hybridize to the labeled probe. The platinum linker labeled nucleotides do hardly interfere with the hybridization, if at all. Also, this technique obviates the use of modified nucleotides in preparing probes.
Nucleotides modified in accordance with the practices of this invention and oligo- and polynucleotides into which the modified nucleotides have been incorporated or oligo- and polynucleotides that have been directly modified using these novel platinum compounds may be used as probes in biomedical research, clinical diagnostics and recombinant DNA technology.
Other advantages and embodiments of the invention will become clear from the following experimental part and the examples.
These compounds, i.e. the linkers having formula (I), may be prepared by a process which involves:
(a) reacting a compound having the structure: 
with KI in a suitable solvent under suitable conditions so as to form a iodated platinum compound having the structure: 
(b) reacting said iodated platinum compound with ethylenediamine in a suitable solvent so as to form a diethyleneamine iodated platinum compound and represented by the formula Pt(en)I2 and having the structure: 
(c) reacting said compound with AgNO3, the reaction being carried out in a suitable solvent, under suitable conditions so as to form a compound having the structure: 
(d) reacting said compound with KCl in a suitable solvent under suitable conditions so as to form a compound having the structure: 
(e) reacting said compound with AgNO3 in a suitable solvent, under suitable conditions so as to form a compound having the structure: 
(f) recovering said compound as modified platinum starting compound for the synthesis of hapten-bound Pt(en) compounds for use as DNA and/or RNA label.